aDepartment of Engineering Science and Mechanics, The Pennsylvania, State
University, University Park, PA, 16802, USA. E-mail: [email protected] and Developmental Biology (CDB) Graduate Program, The Huck Institutes of
the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USAc Department of Food Science, The Pennsylvania State University, University Park, PA,
16802, USAd Ascent Bio-Nano Technologies, Inc., State College, PA, 16802, USAeNational Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA
† Electronic supplementary information (ESI) available: Including videos ofsample enrichment at the entrance and exit of enrichment region, and multi-channel sample enrichment. See DOI: 10.1039/c3lc51001h
Cite this: Lab Chip, 2014, 14, 924
Received 31st August 2013,Accepted 6th December 2013
DOI: 10.1039/c3lc51001h
www.rsc.org/loc
Continuous enrichment of low-abundancecell samples using standing surface acousticwaves (SSAW)†
enrichment regions). Our method is able to concentrate highly diluted blood cells by more than 100 fold
with a recovery efficiency of up to 99%. Such highly effective cell enrichment was achieved without using
sheath flow. The SSAW-based technique presented here is simple, bio-compatible, label-free, and
sheath-flow-free. With these advantages, it could be valuable for many biomedical applications.
Introduction
The ability to enrich cells or other biological samples withhigh viability and recovery efficiency is important in manyapplications in bioanalysis and medical diagnostics.1–7 Thisability is even more critical when dealing with low-abundancecell types (i.e., rare cells), such as circulating tumor cells, stemcells, and fetal cells.8 Enrichment of these low-abundancecells is vital since higher sample concentration often leads tosignificantly improved signal-to-noise ratio in analysis. Con-ventional cell-enrichment techniques, such as centrifugation,are not suitable for use with these low-abundance cell typesdue to the significant loss of cell viability and limited abilityto handle small quantities of cells with high recovery effi-ciency. In this regard, microfluidic-based approaches havesignificant advantages. Unlike the traditional macroscale plat-forms, parameters in microfluidic devices (e.g., channeldimensions, flow profile) can be precisely controlled at thecellular scale; this precise control facilitates high capture
efficiency and isolation purity.9,10 Furthermore, enriched cellscan be quickly and precisely manipulated to next-stage analy-sis (e.g., genomic analysis, drug screening) or on-chip cell cul-turing as part of an integrated and automated process,eliminating the intermediate procedures required in macro-scale systems.11,12
Among all the microfluidic-based cell-enrichment tech-niques, those using non-contact cell trapping are often preferreddue to their ability to limit surface interaction and mechanicalstress on cells and provide convenient sample transfer.13,14 Non-contact cell trapping is usually achieved by applying externalforces to the cells to counteract the viscous drag force in contin-uous flows. Over the past decades, various on-chip techniqueshave been implemented to trap cells or micro-particles inmicrofluidic flows, including dielectrophoresis (DEP),15–17
optical tweezers,18–20 and magnetic tweezers.21,22 Recently,acoustic-based approaches23–29 have received significant atten-tion because of their versatility, bio-compatibility, and label-free, non-contact nature. For example, the standing surfaceacoustic wave (SSAW) based microfluidic techniques haveresulted in a variety of applications (e.g., cell/particle focusing,patterning, separation, and sorting).30–40 Recently, our groupdemonstrated a SSAW-based cell patterning technique thatcan trap cells or microparticles in stationary fluids.41,42 How-ever, SSAW-based cell trapping in moving fluids for on-sitesample enrichment has not yet been demonstrated.
In this work, we demonstrate a SSAW-based cell trappingand enrichment technique that operates in continuous flows.Our experimental results indicate that this SSAW-based cellenrichment technique can achieve a recovery efficiency of more
than 90% for highly diluted blood cells and concentrate thesample by a factor of 100–1000. Experimental results indicateexcellent post-enrichment cell viability. Moreover, detachablemicrochannels enabled by coupling gel allow simultaneousmultichannel processing to enhance the enrichment efficiency.Our SSAW-based device is simple to fabricate and highly flexiblein terms of channel material selection, resulting in significantpotential for system integration and mass production. With itshigh recovery efficiency, bio-compatibility, and simplicity, theSSAW-based cell enrichment approach presented here can bevaluable in many cell-based bioanalytical systems.43–50
Working mechanism
Fig. 1A shows the schematic of the SSAW-based cell enrich-ment device. A pair of parallel interdigital transducers (IDTs)was deposited onto the lithium niobate (LiNbO3) substrate. Amicro-tubing channel with spherical cross-sections was assem-bled in the SSAW-activated region of the substrate with its longaxis oriented in the propagation direction of acoustic waves. Inorder for the acoustic waves to propagate into the microchannel,a coupling gel was used to fill the gap between the tubing andthe substrate, as shown in Fig. 1B. Applying an AC signal to theIDTs results in the generation of a SSAW field and thus the crea-tion of a non-uniform pressure field in the fluid with a periodicdistribution of pressure nodes (i.e., minimum pressure ampli-tude) and antinodes (i.e., maximum pressure amplitude). In thepresence of the SSAW field, a cell suspension was injected intothe microchannel. Upon entering the region where the cou-pling gel bonds the microchannel to the substrate, known as
Fig. 1 (A) Schematic of the SSAW-based sample enrichment device.Two parallel IDTs generate a SSAW field to trap cells inside themicrochannel. (B) Cross-sectional view of the microdevice in the y–zplane. A coupling gel is coated between the tubing and the piezoelec-tric substrate. (C) Mechanism of SSAW-based cell trapping for enrich-ment in continuous flows. The dashed lines indicate the pressuredistribution in the microchannel. The arrows on the cells indicate thedirection of cell movement.
the enrichment region, cells were trapped at SSAW pressurenodes. As more fluid passed through the enrichment region,the concentration of the trapped cells gradually increased untilthe trapping was saturated. Finally, the SSAW was turned off torelease the cells. The enriched sample could be collected fromthe outlet of the microchannel or directly delivered into thedownstream for further analysis and processing.
When cells enter the pressure field, they experience twoforces in the x–y plane: the primary acoustic radiation force(Fr) and the viscous drag force (Fv), which can be expressed as51
Fp V x
rp m
02
24
( , )sin (1)
5 22p m
p m
p
m
(2)
Fv = −6πηrv (3)
where p0, Vp, λ, ϕ, x, ρm, ρp, βm, βp, η, r, and v are pressureamplitude, particle volume, SSAW wavelength, contrast fac-tor, distance from the pressure node, density of medium,density of cells, compressibility of medium, compressibilityof cells, medium viscosity, cell radius, and relative velocity,respectively. As shown in Fig. 1C, the primary radiation forcemoves the particles to the pressure nodes. As the distancebetween particles decreases, the secondary radiation forceplays a dominant role in aggregating particles together andforming an array of clusters.15 The component of the primaryforce along the x axis immobilizes the clusters in the pres-sure nodes by competing with the viscous drag force in theopposite direction. The clusters continue to attract nearbyparticles, growing in size and resulting in an increase in radi-ation forces on clusters. Because each pressure node has amaximum trapping capacity, the saturation occurs graduallyfrom the upstream end to the downstream end of the enrich-ment region. When the volume of a trapped cluster is satu-rated, some particles will be flushed off and trapped again atthe downstream pressure nodes.
Materials and methods
We used Y+128° X-propagation LiNbO3 as a piezoelectric sub-strate to generate SSAW. The IDTs were fabricated throughstandard photolithography processes.32 After depositing ametal double layer (Cr/Au, 50 Å/500Å) with an e-beam evapo-rator (Semicore Corp), two parallel IDTs were formed on theLiNbO3 substrate by a lift-off process. The IDTs we designedhad 20 pairs of electrodes with consistent electrode widthsand spacing gaps (50 μm). Both IDTs could generate identicalsurface acoustic waves (SAWs) with a wavelength of 200 μmat a resonance frequency of 19.6 MHz. Coherent AC signals,which were generated by an RF signal generator (Agilent Tech,
Fig. 2 Recorded fluorescent images at (I) the entrance, (II) the center,and (III) the exit of the enrichment region, respectively. I, II, and IIIindicate the observation locations during enrichment. The whitedashed lines indicate the boundaries of the enrichment region.
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E4422B) and amplified with a power amplifier (AmplifierResearch, 100A250A), were applied to both IDTs to generate twotravelling SAWs along the delay line, forming a one-dimensional(1D) SSAW field. The device was immobilized on the stage ofan inverted microscope (Nikon TE2000U). A CCD camera(CoolSNAP HQ2, Photometrics, Tucson, AZ) was connected tothe microscope to record the cell-enrichment process. Thefluorescence intensity was analyzed with ImageJ 1.46 software.
In the experiments, we used polyethylene tubing (BD,Franklin Lakes, NJ) with an inner diameter of 280 μm as amicrochannel. The micro tubing was assembled at the SAW-activated region of the LiNbO3 substrate with its long axis ori-ented in the direction of SAW propagation (Fig. 1A). The centerof the SSAW-activated region was coated with a KY gel (Johnson& Johnson, New Brunswick, NJ) between the substrate and themicrochannel (Fig. 1B). The coupling length was about 5 mm.A syringe pump (neMESYS, Cetoni GmbH, Korbussen,Germany) was used to control the flow rate. Each sample col-lected from the outlet was subsequently diluted into a propervolume, and counted in a hemacytometer (Hausser Scientific,Horsham, PA) three times. For the highly diluted blood sam-ples (103 cells mL−1), the number of trapped cells was directlycounted in the microchannel through the microscope.
Fluorescent microspheres (Dragon green, 480/520, Bangslaboratories Inc, Fishers, IN) were used to characterize thedevice. Microspheres with a diameter of 7 μm were suspendedin 1% SDS solution at a concentration of 106 particles mL−1.Human whole blood purchased from Zen-bio, Inc. wasdiluted with 1× PBS solution into different concentrations(103–105 cells mL−1) for cell enrichment. Since it was not ableto culture red blood cells, cell viability was tested in a MurineRaw 264.7 macrophage cell line. Cells were either treated bypassing through a microchannel with or without SSAWenrichment, or incubated at 65 °C for 15 min. Cells withoutany treatment and culture medium were used as a positiveand a negative control, respectively. After treatment, cells wereseeded in a 96-well plate in a complete Dulbecco's modifiedEagle's medium (DMEM, Invitrogen) containing 10% (v/v)fetal bovine serum (Atlanta Biologicals) and 1% (v/v) penicillin–streptomycin (Cellgro) at a density of 105 cells mL−1. After24 h, cell viability was determined by MTT assay52 and itsabsorbance at 450 nm was expressed as mean ± standarddeviation of five experimental measurements.
Results and discussionSSAW-based particle trapping
The coupling gel between the microchannel and piezoelectricsubstrate allows acoustic waves to propagate into the fluidsin the microchannel and generate a non-uniform pressuredistribution. In our design, the length of the enrichmentregion is about 5 mm and the distance between each adja-cent pressure node is 100 μm (half wavelength). The numberof pressure nodes (trapping positions) created in themicrochannel is approximately 50, providing a high trapping
926 | Lab Chip, 2014, 14, 924–930
capacity. Fig. 2 shows the fluorescent images of the particlestrapped along different locations of the enrichment region. Inthe entrance of enrichment region (Fig. 2-I), a comparisonbetween the air medium (non-enrichment region) and the gelmedium (enrichment region) is presented. After the SSAWfield was applied, fluorescent polystyrene beads flowed intothe trapping region and aggregated at the pressure nodes. Theparticles which had not entered the enrichment region experi-enced no acoustic radiation force and remained randomly dis-tributed in the microchannel. Fig. 2-II shows that particlesplaced in the middle of the enrichment region were well pat-terned by the acoustic radiation forces. While the power wason, all of the particles remained inside the enrichment regionas shown in Fig. 2-III. Even though continuous flows wereapplied, no particle was observed in the lower reaches of themicrochannel (outside the trapping region), indicating effec-tive particle trapping by SSAW (see ESI† Video 1).
Device characterization
We used fluorescent beads to characterize the process ofsample enrichment. As illustrated in Fig. 3A, this processhas three stages: enrichment, saturation, and release. Fig. 3Bshows typical images of each stage (see ESI† Video 2 and
Fig. 3 (A) The sample enrichment process presented as variation in the fluorescence intensity. I–VI indicate six specific points of time during theprocess. (B) Recorded fluorescent images of the enrichment-region entrance at the points of time (I–VI) indicated in (A). The influence of different (C)input powers and (D) flow rates on SSAW-based sample enrichment. The error bars denote the standard deviation of three experimental measurements.
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Video 3). Before SSAW was turned on, particles passedthrough the detection region at a consistent velocity (Fig. 3B-I),resulting in little change of the fluorescence intensity. Theintensity started to increase as soon as a SSAW field wasapplied. The growth rate was approximately linear becausethe acoustic radiation force captured most of the particlesonce they entered the trapping region. During this period(the enrichment stage), the sample was gradually enrichedinside the enrichment region (Fig. 3B-II and III). When thetrapped sample approached the maximum capacity of thepressure node, some trapped particles were released and newparticles were trapped achieving dynamic equilibrium andretaining a constant amount of particles in the pressure node(Fig. 3B-IV and V). This saturation stage started with a sharpdecrease in the growth rate of the fluorescence intensity. Dur-ing saturation, the intensity was maintained at a certain level(~150 in this case) with small fluctuations, as shown in Fig. 3A.The stable immobilization of sample inside the microchannelallowed for the introduction of a washing buffer as an addi-tional purification step. The purification step is useful toremove contaminated molecules and exchange mediums, animportant process in online biochemical analysis.22 As soonas the SSAW was turned off, the fluorescence intensity experi-enced an immediate jump (a sharp peak in Fig. 3A) since alarge amount of the fluorescent beads were released andsimultaneously passed through the detection region. The
particles were removed by the flow (Fig. 3B-VI), leading to afast decay of the fluorescence intensity.
We further studied the influence of input power onenrichment ability, as shown in Fig. 3C. Four power levelsfrom 16 to 19 dBm were used to enrich the particles at asingle flow rate. When the input power was tuned from 16 to18 dBm, the fluorescence intensity at the saturation stageincreased from ~50 to ~150, indicating that a higher trappingcapacity was achieved due to stronger acoustic radiationforces. Though a higher input power resulted in a higherpressure amplitude (p0) and thus stronger acoustic radiationforces exerted on the particles according to eqn (1), acousticstreaming also became obvious at higher power levels(19 dBm and above).53 Fluorescence intensity grew linearlyduring the first 70 s but then suddenly dropped to the origi-nal level in the next 10 s. This was because the acousticstreaming became dominant and particles failed to remaintrapped at the pressure nodes.
The flow rate is another factor that influences the sampleenrichment. To trap particles at the pressure nodes, theacoustic radiation forces (Fr) should be larger than the viscousdrag forces (Fv). A higher flow rate could help to enhancethroughput, but also exerts larger viscous drag forces on parti-cles due to the higher particle velocity (v) in eqn (3), leadingto a decrease in the recovery efficiency. We conducted sampleenrichment with four different flow rates and a constant input
power of 18 dBm. As shown in Fig. 3D, a flow rate of 5 μL min−1
achieved a smaller growth rate in fluorescence intensity than10 μL min−1, since the smaller flow velocity of the particlesinduced a lower speed of sample accumulation. However,when flow rates of 15 and 20 μL min−1 were used, the satu-rated fluorescence intensity decreased by approximately 42%as compared to the flow rate of 10 μL min−1. At a higher flowrate, the stronger viscous drag forces enable particles toescape trapping by acoustic radiation forces.
Parallel sample enrichment
Polydimethylsiloxane (PDMS) has been widely used in SAWmicrofluidics for the fabrication of predesigned microchannels,which can then be bonded to a piezoelectric substrate to forma SSAW-based microdevice. However, the relatively high costof the piezoelectric substrate limits the device's utility in dis-posable applications. In our work, polyethylene micro tubingswere assembled onto the LiNbO3 substrate as microchannels.After each run, the used tubings could be easily peeled offand replaced by new tubings to avoid cross-contamination. Inaddition to disposability, the micro tubings enable a multi-channel, SSAW-based sample enrichment configuration withour device. The parallel sample enrichment could help toimprove the working efficiency and throughput by enrichingmore cells in less time, or enriching different species ofcells simultaneously. As shown in Fig. 4A, by assembling anarray of micro-tubing channels (three in this case) ontothe piezoelectric substrate, parallel sample enrichment was
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Fig. 4 (A) SSAW-based parallel sample enrichment by assembling multi-ple micro-tubing channels onto the piezoelectric substrate. Right laneshows the fluorescent image of simultaneous sample enrichment in threemicrochannels (see ESI† Video 4). (B) Performance of parallel sampleenrichment in three microchannels presented as fluorescence intensity.
conveniently established. In each channel, the efficiency ofthe sample enrichment was consistent (Fig. 4B), revealing asteady performance of each working unit.
Enrichment of highly diluted blood cells
Serially diluted human whole blood was used as a sample forcell enrichment to demonstrate the capability of our device. Bloodcells with three concentrations (105, 104, and 103 cells mL−1)were studied in the experiments. Each sample was enrichedfor 10 min at a flow rate of 7 μL min−1. The enrichment pro-cess is shown in Fig. 5A. The blood cells gradually accumu-lated at the pressure nodes once they entered the enrichmentregion, resulting in growth of cell clusters. Since the cell sam-ples were highly diluted, trapping saturation did not occurduring the enrichment process. The recovery efficiency forthe enrichment of diluted blood cells was calculated andshown in Fig. 5B. The recovery efficiency was defined as theratio of measured cell concentration to the theoretical 100%-recovered concentration. A recovery efficiency of 93.1 ± 2.9%and 97.3 ± 5.2% was obtained when diluting the blood sampleto 105 cells mL−1 and 104 cells mL−1, respectively. Whenthe blood sample was further diluted to a concentration of103 cells mL−1, 99.1 ± 11.3% of the blood cells could be recov-ered. Since the volume of the enrichment region was about0.3 μL (5 mm in length, 0.28 mm in diameter), after runningfor 10 min with a flow rate of 7 μL min−1, 70 μL of the samplewas enriched into a final volume of 0.3 μL with a recovery effi-ciency more than 90%, indicating that the sample was concen-trated by two orders of magnitude. The concentration factor
Fig. 5 (A) i–iii: Dynamic process of blood cell enrichment. The imageswere taken in 200 s interval for 600 s. iv: Magnified image of cellclusters. (B) Recovery efficiency of cell enrichment at differentconcentrations. The error bars denote the standard deviation of threeexperimental measurements.
could be further increased with an increase in processedsample volume. As a result, the concentration of the sampleafter enrichment could be enhanced up to 100–1000 times,which shows an improvement over many other cell or par-ticle enrichment techniques (typical concentration factor:<100 times).54–60
Cell viability test
One of the biggest advantages of our SSAW-based cell enrich-ment technique is its bio-compatibility. In this regard, wemeasured the viability of cells after SSAW enrichment byMTT assay. Cells without any treatment and culture mediumwere used as a positive and a negative control, respectively.As shown in Fig. 6, the viability of cells which passed throughthe channel with and without SSAW enrichment was mea-sured to be at the same level as the positive control (between0.25 and 0.30), while the viability of the cells incubated at65 °C for 15 min was close to the value of culture medium(lower than 0.10), indicating that the process of SSAW-basedenrichment had no significant effect on cell viability. Thoughthe viabilities of cells passing through the channel with andwithout SSAW enrichment (the second and third bars inFig. 6) were observed to be different, the P values betweenthese two groups and the positive control (the first bar) werecalculated to be 0.9204 and 0.3108, both of which are largerthan 0.05. As a result, the difference of these three groupscould be considered to be insignificant.
Fig. 6 Cell viability test. Murine Raw 264.7 macrophages were eithertreated by passing through a microchannel with or without SSAWenrichment, or incubated at 65 °C for 15 min. Cells without anytreatment and culture medium were used as a positive and a negativecontrol, respectively. After treatment, cells were seeded in a 96-well platefor 24 h, and cell viability was determined by MTT assay and expressed asmean ± standard deviation of three experimental measurements.
Conclusions
By applying a SSAW field to induce a non-uniform pressuredistribution in the microfluidic channel, a large number ofpressure nodes can be generated to trap cells for sampleenrichment. This SSAW-based technique allows the enrich-ment of highly-diluted cell samples with excellent perfor-mance (concentration factor: 100–1000; recovery efficiency:over 90%; high viability). In addition, the gel-coupling methodprovides further advantages: flexibility in the channel mate-rials, simple fabrication, and high-throughput parallel sampleenrichment. These advantages make the SSAW-based tech-nique presented here promising in the enrichment of low-abundance rare cells for cellular study.
Acknowledgements
We gratefully acknowledge financial support from NationalInstitutes of Health (Director's New Innovator Award,1DP2OD007209-01), American Asthma Foundation (AAF)Scholar Award, the National Science Foundation and thePenn State Center for Nanoscale Science (MRSEC) undergrant DMR-0820404. J.P.M. and S.J.L. are supported by theNHLBI Division of Intramural Research. Components ofthis work were conducted at the Penn State node of theNSF-funded National Nanotechnology Infrastructure Network.
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